The present invention is related with the field of biotechnology and pharmaceutical industry, in particular with active immunization employing as targets molecules related with angiogenesis.
The process of formation of new blood vessels from pre-existent ones is called angiogenesis. This event is widely regulated through the equilibrium of pro- and anti-angiogenic factors. Among the diseases in which the course has been related with the induction of pro-angiogenic factors and the formation of new blood vessels in anomalous form are: (a) cancer (both primary tumors and their metastases), (b) acute and chronic inflammatory processes such as asthma, respiratory distress, endometriosis, atherosclerosis, and tissular edema, (c) diseases of infectious origin as the Hepatitis, and Kaposi sarcoma, (d) autoimmune diseases as diabetes, psoriasis, rheumatoid arthritis, thyroiditis, and (e) other diseases and states as the diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma, and angiofibroma (Carmelliet P. y Jain R K. Nature 407:249, 2000; Kuwano M, et al. Intern Med 40:565, 2001). A potentially attractive therapeutic procedure for many of these cases could be based on the inhibition of the activity of the pro-angiogenic factors, that stimulate the anomalous formation of blood vessels, via their neutralization, or that of their receptors, or by eliminating the sources that produces them.
Vascular endothelium growth factors are a family of molecules that induce the formation of new vessels specifically and directly (Leung Science 246:1306, 1989; Klagsburn M, Annual Rev Physiol 33:217, 1991). This family includes the vascular permeability factor, also known as vascular endothelium growth factor VPF/VEGF (now denominated VEGF-A), the placenta growth factor PlGF, the platelet derived growth factors PDGF-A and PDGF-B, and other four new molecules structurally and functionally related to VEGF-A designated VEGF-B/VRF, VEGF-C/VRP, VEGD-D/FIGF, and VEGF-E. (Olofsson B et al. PNAS USA 13:2576, 1996; Joukov V et al. EMBO J 15:290, 1996; Yamada Y et al. Genomics 42:483, 1997; Ogawa S et al. J Biol Chem 273:31273, 1998).
VEGF-A is a homodimeric glycoprotein formed by two 23-kDa subunits (Ferrara N, et al. Biochem Biophys Res Comun 165:198, 1989), of which five monomeric isoforms exist, derived from the differential splicing of the same RNA. These include two isoforms that remain attached to the cellular membrane (VEGF 189 and VEGF 206), and three of soluble nature (VEGF 121, VEGF 145, and VEGF 165). VEGF 165 is the most abundant isoform in mammal tissues, except for lung and heart, where VEGF 189 predominates (Neufeld G et al. Canc Met Rev 15:153, 1995), and in placenta, where VEGF 121 expression prevails (Shibuya M A et al. Adv Canc Res 67:281, 1995).
VEGF-A is the most studied and characterized protein of this family, and its alteration has been described in a larger number of diseases. Its over-expression is associated to tumors of different origin and localization, and their metastasis (Grunstein J et al. Cancer Res 59:1592, 1999), chronic inflammatory processes as ulcerative colitis and Crohn's disease (Kanazawa S, et al. Am J Gastroenterol 96:822, 2001), psoriasis (Detmar M, et al. J Exp Med 180:1141, 1994), respiratory distress (Thickett D R et al. Am J Respir Crit Care Med 164:1601, 2001), atherosclerosis (Celletti F L et al. Nat Med 7:425, 2001; Couffinhal T et al. Am J Pathol 150:1653, 1997), endometriosis (McLaren J. Hum Reprod Update 6:45, 200), asthma (Hoshino M, et al. J Allergy Clin Immunol 107:295, 2001), rheumatoid arthritis and osteoarthritis (Pufe T et al. J Rheumatol 28:1482, 2001), thyroiditis (Nagura S et al. Hum Pathol 32:10, 2001), diabetic and newborn retinopathies (Murata T et al. Lab Invest 74:819, 1996; Reynolds J D. Paediatr Drugs 3:263, 2001), macular degeneration and glaucoma (Wells J A et al. Br J Ophthalmol 80:363, 1996; Tripathi R C et al. Ophthalmology 105:232, 1998), tissular edema (Kaner R J et al Am J Respir Cell Mol Biol. 22:640 2000; Ferrara N Endocrinol Rev 13:18, 1992), obesity (Tonello C et al. FEBS Lett 442:167, 1999), hemangiomas (Wizigmann S y Plate K H Histol Histopathol 11:1049, 1996), in the synovial fluid of patients with inflammatory arthropathies (Bottomley M J et al Clin Exp Immunol 119:182, 2000), and associated to transplant rejection (Vasir B, et al. Transplantation 71:924, 2001). In the particular case of tumors, the cells expressing the three basic isoforms of VEGF-A: 121, 165, and 189, are the ones that grow faster in vivo; while in final stages most tumors limit expression to the VEGF 165 isoform, or, in its absence, to a combination of 121 and 189 that far from being additive, evidences a cooperation that strengthens the tumor vascular network (Grunstein J. Mol Cell Biol 20:7282, 2000).
PlGF, described in 1991, is not able to induce endothelial proliferation in its homodimeric form (Maglione D et al. Proc Natl Acad Sci USA 88:9267, 1991, DiSalvo J et al. J Biol Chem 270:7717, 1995). With PlGF up-regulation, and with it, of the signal transduced via VEGFR-1, the endothelial cells amplify their responses to VEGF during the change to the angiogenic phenotype associated to certain pathologies (Carmeliet P et al. Nat Med 7:575, 2001). PlGF expression has been related to the vascularization of human meningioma and glioma (Nomura M et al. J Neurooncol 40:123, 1998). This molecule forms heterodimers with VEGF 165, with pro-angiogenic activity, and their over-expression has been described in the conditioned media of some tumor cell lines (Cao Y et al. J Biol Chem 271:3154, 1996), and associated to the evolution of rheumatoid arthritis and to primary inflammatory arthropathies, in general (Bottomley M J et al. Clin Exp Immunol 119:182, 2000).
The over-expression of the rest of the members of the VEGF family, less studied, is also associated to a number of pathologies. VEGF-B has been related to breast, ovary, and kidney tumors, and to melanomas and fibrosarcomas (Sowter H M, et al. Lab. Invest. 77:607, 1997; Salven P Am. J. Pathol. 153:103, 1998, Gunningham S P et al. Cancer Res 61:3206, 2001). The differential expression of the VEGF-B 167 isoform in vitro has been reported in tumor cells of diverse origin (Li X, et al. Growth Factors 19:49, 2001). VEGF-C and VEGF-D are involved in the regulation of lymphatic vessels formation (Joukov V. et al EMBO J. 15: 290, 1996), and VEGF-C over-expression is associated to tissular edemas, to tumors of the breast, lung, head and neck, esophagus, and stomach, lymphomas, prostate cancer, and metastatic nodes (Kajita T, et al. Br J Cancer 85:255, 2001; Kitadi Y, et al Int J Cancer 93:662, 2001; Hashimoto I, et al. Br J Cancer 85:93, 2001; Kinoshita J, et al. Breast Cancer Res Treat 66:159, 2001; Ueda M, et al. Gynecol Oncol 82:162, 2001; Salven P Am. J. Pathol. 153:103, 1998; O-Charoenrat P et al. Cancer 92:556, 2001). In the case of VEGF-D, its over-expression by tumor cells is related to an in vivo increase of lymphatic vasculature in the tumors and the increase of metastasis in lymphatic nodes (Stacker S A, et al. Nat Med 7:186, 2001; Marconcini L et al. Proc Natl Acad Sci USA 96:9671, 1999).
The alterations on endothelial cell function induced by the molecules of the VEGF family are mediated by their binding to cell receptors of the type tyrosine kinase class 3, that so far include: VEGFR1 (Flt1), VEGFR2 (KDR/Flk1), and VEGFR3 (Flt4) (Kaipainen A J. Exp. Med. 178:2077, 1993). The N-terminal domain 2 has been identified as responsible of the binding to the ligands, favoring the phosphorilation of the cytoplasmatic domain and transduction of the signal (Davis-Smyth T et al EMBO 15:4919, 1996).
Ligands identified for VEGFR1 include VEGF-A, PlGF, and VEGF-B, in decreasing order of affinity (Shibuya M Int J Biochem Cell Biol 33: 409, 2001). In endothelial cells, this receptor captures the circulating VEGF (Gille H et al EMBO J. 19:4064, 2000). The binding of VEGF-A to the VEGFR1 expressed in cells of the hematopoyetic lineage affects significantly the activation of transcriptional factor NFκB in the precursors of dendritic cells, and in B and T lymphocytes. This last interaction is relevant in the in vivo establishment of an unfavorable immunologic balance, where dendritic cells maturation and the fraction of T lymphocytes are reduced, a phenomenon observed on immunosupressed patients and in particular, with cancer (Dikov M M et al Canc Res 61:2015, 2001; Gabrilovich D et al. Blood 92:4150, 1998). Over-expression of VEGFR1 has been related with psoriasis, endometrial cancer and hepatocellular carcinoma (Detmar M, et al. J Exp Med 180:1141, 1994; Ng I O Am J Clin Patol 116:838, 2001; Yokoyama Y et al Gynecol Oncol 77:413, 2000).
The VEGFR2 receptor (KDR/Flk1) mediates the biological effects of VEGF-A, and also binds VEGF-C and VEGF-D. This receptor is expressed differentially on activated endothelium and in some cell lines of tumor origin where it establishes autocrine pathways with the secreted VEGF. Apart from being involved in the already mentioned pathologies that are related with the over-expression of its ligands, the over-expression of VEGFR2 has been related with the progression of endometrial cancer (Giatromanolaki A et al, Cancer 92:2569, 2001), malignant mesothelioma (Strizzi L et al. J Pathol 193:468, 2001), astrocytic neoplasms (Carroll R S et al. Cancer 86:1335, 1999), primary breast cancer (Kranz A et al. Int J Cancer 84:293, 1999), intestinal type gastric cancer (Takahashi Y et al Clin Cancer Res 2:1679, 1996), multiform glioblastoma, anaplastic oligodendroglioma, and necrotic ependimoma (Chan A S et al. Am J Surg Pathol 22:816, 1998). Over-expression of KDR has also been associated to the autosomic disease VHL and to hemangioblastoma (Wizigmann-Voos S et al Cancer Res 55:1358, 1995), to the progression of diabetic retinopathy (Ishibashi T. Jpn J Ophthalmol 44:323. 2000) and, in combination with Flt-1 over-expression, to the delayed-type hypersensitivity reactions (Brown L F et al J Immunol 154:2801, 1995).
Lymphangiogenesis mediated by VEGF-C and VEGF-D results from their binding to the FLT4 receptor or VEGFR3, expressed in the lymphatic endothelium. In some cases, even when over-expression of the ligands is not present, the over-expression of the receptor has been related to an adverse prognosis in the course of a group of pathologic entities, including: diabetic retinopathy (Smith G. Br J Ophthalmol 1999 April; 83(4):486-94), chronic inflammation and ulcers (Paavonen K et al, Am J Pathol 156:1499, 2000), the establishment of metastasis in lymphatic nodes and progression of breast cancer (Gunningham S P. Clin Cancer Res 6:4278, 2000 Valtola R et al. Am J Pathol 154:1381, 1999), associated to nasopharyngeal tumors and squamous oral carcinomas (Saaristo A et al. Am J Pathol 157:7, 2000; Moriyama M et al. Oral Oncol 33:369, 1997). Moreover, the over-expression of VEGFR3 is a sensitive marker of Kaposi sarcoma, type Dabska hemangioendothelioma and of cutaneous lymphangiomatosis (Folpe A L et al. Mod Pathol 13:180, 2000; Lymboussaki A et al. Am J Pathol 153:395, 1998).
Recently, two receptors were identified for VEGF named NRP1 and NRP2. These belong to the neurophilins family (NRP), and act as co-receptors for some specific isoforms of proteins of the VEGF family: VEGF-A145 VEGF-A165, VEGF-B167 and PlGF1, increasing their mitogenic capacity. The expression of NRP1 has become a marker of the aggressiveness of prostate cancer, has been related to the increase of angiogenesis in melanomas, and with apoptosis escape events in breast cancer (Latil A et al. Int J Cancer 89:167, 2000; Lacal P M J Invest Dermatol 115:1000, 2000; Bachelder R E Cancer Res 61:5736, 2001). The coordinate over-expression of NRP1, KDR, and VEGF-A165 have been related to the fibrovascular proliferation in diabetic retinopathy cases and rheumatoid arthritis (Ishida S. et al. Invest Ophthalmol V is Sci 41: 1649, 2000; Ikeda M. Et al. J Pathol 191:426, 2000). NRP2 is over-expressed in osteosarcomas where it promotes angiogenesis and tumor growth (Handa A et al. Int J Oncol 17:291, 2000).
Most of the therapeutic strategies based on angiogenesis inhibition, especially in cancer treatment, are based in the blockade of molecules of the VEGF family and their receptors, with clinical trials in course using: (1) monoclonal antibodies blocking VEGF or the KDR receptor, (2) metalloproteinase inhibitors, as Neovastat and Prinomastat, (3) VEGF inhibitors as Thalidomide, Suramin, Troponin I, and IFN-α and Neovastat, (4) blockers of VEGF receptors as SU5416, FTK787 and SU6668, (5) inducers of tumor endothelium apoptosis, as Endostatin and CA4-P, and (6) ribozymes that decrease VEGF or VEGF receptors expression (Angiozyme). Due to the high homology between human VEGF and its receptors KDR and Flt-1 with their murine homologs (˜90%, 81%, and 89%, respectively), many animal models are used routinely to evaluate the preclinical effectiveness of antiangiogenic compounds directed to this system (Hicklin D J et al. DDT 6:517, 2001).
Passive administration of antibodies to VEGF or VEGFRs is successfully tested in different clinical phases in humans (Hicklin D J et al. DDT 6:517, 2001). The anti-VEGF humanized monoclonal antibody A.4.6.1 (Genentech, San Francisco, United States) is in phase III clinical trial for the treatment of colon, breast, kidney, and lung tumors (Kim, K J et al. Nature 362:841, 1993; Boersig C. R&D Directions October 7:44, 2001). In particular, for the case of the KDR receptor, a monoclonal antibody has been developed (IMC-1C11, ImClone) that recognizes the N-terminal extracellular domain of this receptor, and inhibits proliferation and migration of leukemic human cells, increasing survival of xenotransplanted mice. At present, its effect is being studied in patients with colon cancer metastasis (Dias S et al. J Clin Invest 106:511, 2000). In the aforementioned trials, the absence of concomitant adverse effects with the application of these monoclonal antibodies has been demonstrated.
Notwithstanding the previous, a therapeutic modality not yet employed for the blockade of neoangionegesis is specific active immunotherapy (SAI). In the SAI of cancer, antigens as peptides, proteins or DNA are employed, mixed with appropriate adjuvants. SAI procedures pursue the stimulation of an immune response, both of the humoral (activation of B-lymphocytes), and cellular types (activation of T helper, and cytotoxic lymphocytes, and natural killer cells), associated to dendritic cell function as professional presenting cells in the MCHI and MHC II contexts (Bystryn J C, Medscape Hematology-Oncology 4:1, 2001; Parker, K C et al., J. Immunol 152:163, 1994; Nestle F O et al., Nature Medicine 7:761, 2001; Timmerman J M, Annual Review Medicine 50:507, 1999).
SAI is a rapidly growing field of experimental and clinical research, with attractive applications, especially in oncology, where more than 60 undergoing clinical trials based in procedures of SAI are reported, which surpass at present the clinical trials based on the use of monoclonal antibodies. In the particular case of cancer, the antigens used as immunogens for SAI are selected because of their physiological relevance and difficulty of being substituted in the processes of tumor phenotypic drift (Bodey B et al., Anticancer Research 20: 2665, 2000), and because of their high specific association with the growth and evolution of tumor tissues.
The strategy of treating cancer using SAI also considers preferably the identification of antigens expressed in different tumor types, what could increase the number of indications for the same vaccine preparation. Examples of these are carcinoembryonic antigen (CEA), HER2-neu, human telomerase, and gangliosides (Greener M., Mol Med Today 6:257 2000; Rice J, et al. J Immunol 167:1558, 2001; Carr A et al, Melanoma Res 11:219, 2001; Murray J L, et al. Semin Oncol 27:71, 2000).
In human tumors, VEGF is over-expressed in the tumor compartment (Ferrara, N. Curr. Top. Microbiol. Immunol. 237:1, 1999), and high levels of VEGF and its receptors have been demonstrated in the tumor-associated vasculature (Brekken R A. J Control Release 74:173, 2001). The stromal cells also produce VEGF in response to the stimulus of transformed cells, with the result that when tumor cells are removed, VEGF levels persist in the patients. The presence of VEGF and its receptors have a practical value for the establishment of prognosis and staging in cases of prostate, cervix, and breast tumors (George D J et al. Clin Cancer Res 7:1932, 2001; Dobbs S P et al. Br J Cancer 76:1410, 1997; Callagy G et al. Appl Immunohistochem Mol Morphol 8:104, 2000). On the other hand, VEGF is also within the group of soluble factors that, together with other cytokines like IL-10, TNF-α and TGF-β, (Ohm J E y Carbone D P, Immunol Res 23:263, 2001), could be implicated in the immunosuppression that characterizes cancer patients (Staveley K, et al. Proc Natl Acad Sci USA 95:1178, 1998; Lee K H, et al. J Immunol 161:4183, 1998). This immunosuppressive effect seems to be related to its binding to the Flt1 receptor (Gabrilovich D et al. Blood 92:4150, 1998).
The present invention describes procedures of SAI in experimental tumors using molecules of the VEGF family and their receptors. The antitumoral effects obtained could be based in at least four different mechanisms, without discarding their possible combinations: (a) direct destruction of cancer and stromal cells producing VEGF, by cytotoxic lymphocytes, (b) damaging of endothelial cells of tumor-associated vessels due to the capture or neutralization of the circulating VEGF via antibodies, (c) direct destruction of endothelial cells that express VEGF receptors, by cytotoxic lymphocytes or complement fixing antibodies, (d) activation of a local immune response as a consequence of the capture or neutralization of circulating VEGF, and its consequent elimination of its immunosuppressive effects.
Ideally, these treatments could be used to diminish or avoid the appearance of metastasis, to reduce or eliminate primary tumors as a first or second line therapy, in combination or not with other anti-tumor agents.
Active immunization directed to VEGF family and their receptors could also be efficient in the single or combined therapy of acute and chronic inflammatory processes (asthma, respiratory distress, endometriosis, atherosclerosis, tissular edema), infectious diseases (Hepatitis, Kaposi sarcoma), autoimmune diseases (diabetes, psoriasis, rheumatoid arthritis, thyroiditis, synovitis), diabetic and newborn retinopathies, organ transplant rejection, macular degeneration, neovascular glaucoma, hemangioma, and angiofibroma, among others.